Homeostasis, proliferation, and differentiation in mammalian cells are regulated by the complex circuitry of interacting proteins. Perturbation of these interactions can lead to disease states such as cancer. Thus, analyzing protein-protein interactions is of extreme importance to understanding metazoan physiology.
Protein-protein interactions are involved in every cellular process ranging from gene expression and signal transduction to cell division and differentiation, yet they have been among some of the most difficult aspects of cell biology. Standard biochemical methods have yielded most of the available information about such interactions, but these assays are often limited by the available reagents such as monoclonal antibodies for immunoprecipitation, or lack of appropriate cellular context.
The development of fusion-protein based assays, such as the yeast two-hybrid method (Fields, S. & Song, O. (1989) Nature 340, 245-6.), have greatly expanded the potential for studying protein interactions in intact cells. However, this assay relies on the transcription of a reporter gene; consequently it is not applicable to studies of the kinetics of protein-protein interactions and is unable to detect the interaction of compartmentalized proteins such as receptors at the cell surface. A method based on fluorescence resonance energy transfer (FRET) provided a further advance and is currently one of the most accurate methods used to monitor dynamic interactions (Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S. & Tsien, R. Y. (1991) Nature 349, 694-7.). However, the incremental changes in fluorescence assayed by FRET are small and the stringent steric requirements for detecting the interacting proteins can restrict the utility of this technique.
Assays based on the complementation of enzyme fragments fused to interacting proteins that regenerate enzymatic activity upon dimerization are particularly well suited to monitoring inducible protein interactions (reviewed in Rossi, F. M., Blakely, B. T. & Blau, H. M. (2000) Trends Cell Biol. 10, 119-122). These systems have important advantages including low level expression of the test proteins, generation of signal as a direct result of the interaction, and enzymatic amplification. As a result, they are highly sensitive and physiologically relevant assays (Blakely, B. T., Rossi, F. M., Tillotson, B., Palmer, M., Estelles, A. & Blau, H. M. (2000) Nature Biotechnol. 18, 218-22). Additionally, assays based on enzyme complementation can be performed in any cell type of interest or in diverse cellular compartments such as the nucleus, secretory vesicles or plasma membrane.
Systems for the study of protein-protein interactions have been described which utilize two fusion genes whose products reconstitute the function of a transcriptional activator. Fields et al., (1989) Nature 340:245-247; Bai et al., (1996) Meth. Enzymol. 273:331-347; Luo et al., (1997) BioTechniques 22(2):350-352. In one fusion gene, a sequence encoding a first protein is conjugated to a sequence encoding a DNA-binding domain of a transcriptional regulatory protein. In a second fusion gene, a sequence encoding a second protein is conjugated to a sequence encoding a transcriptional activation domain of a transcriptional regulatory protein. The two fusion genes are co-transfected into a cell which also contains a reporter gene whose expression is controlled by a DNA regulatory sequence that is bound by the DNA-binding domain encoded by the first fusion gene. Expression of the reporter gene requires that a transcriptional activation domain be brought adjacent to the DNA regulatory sequence. Binding of the first protein to the second protein will bring the transcriptional activation domain encoded by the second fusion gene into proximity with the DNA-binding domain encoded by the first fusion gene, thereby stimulating transcription of the reporter gene. Thus, the level of expression of the reporter gene will reflect the degree of binding between the first and second proteins.
There are several disadvantages associated with the use of the above-mentioned system. As it is dependent upon transcriptionally-regulated expression of a reporter gene, this system is limited to the assay of interactions that take place in the nucleus. In addition, the assay is indirect, relying on transcriptional activation of a reporter gene whose product is diffusible. Hence, a method which would allow a direct and immediate examination of molecular interactions, at the site where they occur, would be desirable.
A system for detecting protein-protein interactions, not limited to nuclear interactions, has been described in U.S. Pat. Nos. 5,503,977 and 5,585,245. In this system, fusions between potential interacting polypeptides and mutant subunits of the protein Ubiquitin are formed. Juxtaposition of the two Ubiquitin subunits brought about by interaction between potential interacting polypeptides which creates a substrate for a Ubiquitin-specific protease, and a small peptide reporter fragment is released. In this system, binding between the potential interacting polypeptides does not generate any type of enzymatic activity. Therefore, signal amplification is not possible. Additionally, the ubiquitin system does not measure in situ activity in intact cells, but relies on assays of proteolysis in cell-free extracts. What is needed is a sensitive method for examining protein interactions in intact cells in the relevant cellular compartment.
The possibility of enzyme fragment complementation with beta-galactosidase (β-gal) was first shown in prokaryotes. (Ullman, A. et al. J. Mol. Biol. 24, 339-343 (1967); Ullman, A. et al. J. Mol. Biol 32, 1-13 (1968); Ullman, A. et al. J. Mol. Biol. 12, 918-923 (1965)). Later studies furthered this technology by extending β-gal complementation to mammalian cells and showing that it could be used to monitor inducible protein-protein interactions such as high affinity rapamycin binding proteins and epidermal growth factor (EGF) receptor dimerization. (Mohler, W. & Blau, H. (1996) Proc. Natl. Acad. Sci. USA 93, 12423-12427; Rossi, F., Charlton, C. & Blau, H. (1997) Proc. Natl. Acad. Sci. USA 94, 8405-8410; Blakely, B. et al. (2000) Nat Biotechnol 18, 218-222). U.S. Pat. No. 6,342,345 (Blau, et al.) discloses a enzyme fragment complementation system using beta-galactosidase (β-gal). An alternative complementation system utilized dihydrofolate reductase (DHFR) fragments to study erythropoietin receptor dimerization. (Remy, I. et al. (1999) Science 283, 990-993; Remy, I. & Michnick, S. (1999) Proc. Natl. Acad. Sci. USA 96, 5394-5399).
However, both DHFR and β-gal fragment complementation have their limitations. DHFR fragment complementation is measured by growth, where approximately 25 reconstituted DHFR molecules are required for cell survival. Remy, I. et al. (1999) Science 283, 990-993. Thus, the assay does not directly monitor real-time protein-protein interactions. Moreover, the DHFR interaction is stoichiometric and does not benefit from enzymatic amplification of the signal. Consequently, the signal is weak or requires significant overexpression of the fusion proteins. In addition, mammalian cells have endogenous DHFR, which may increase the background levels of enzyme activity.
The β-gal complementation system of U.S. Pat. No. 6,342,345 (Blau, et al.) and as described in the literature enzymatically amplifies of the signal and can be used to monitor interactions in live cells in real-time. (Rossi, F., Charlton, C. & Blau, H. (1997) Proc. Natl. Acad. Sci. USA 94, 8405-8410; Blakely, B. et al. (2000) Nat Biotechnol 18, 218-222). However, β-gal is a large 90 kD molecule which may sterically hinder the same interaction it seeks to monitor. In addition to the large size of the subunits, β-gal also has the disadvantage of being a tetrameric complex. The need to form a multimeric complex detracts from the usefulness of this system. β-gal also lacks a cell permeable substrate. Hypotonic shock, used to introduce the β-gal substrate into cells, is not ideal because it can affect substrate localization within the cell and can limit the amount of available substrate due to osmotic constraints.
What is desired is a complementation system that utilizes a small protein which has enzymatic activity to allow for signal amplification and a cell permeable substrate.